The present invention is concerned with a process and a reagent kit for the specific determination of the serum fructosamine content in blood or in samples obtained from blood in which disturbing sample components are removed before measurement of the fructosamine content.
The serum fructosamine content is to be understood to mean the total content of non-enzymatically glycosylated serum proteins. These arise when serum glucose forms Schiff's bases via its carbonyl group with free protein amino residues which subsequently, by means of an Amadori rearrangement, are converted into fructosamines with a stable ketoamine bond.
Because of the stability of the ketoamine bond, the half-life time of the serum fructosamine is practically identical with that of the serum proteins, the half life of which is, on average, about 21 days. Corresponding investigations have been published by L. Y. Seng and M. J. Staley in J. Clin. Chem. 32, 560 (1986).
The extent of the fructosamine formation is proportional to the blood glucose level. As is known in the case of diabetics with insufficient dietetic and medicamentary metabolic adjustment, this can be subject to considerable variation, coupled with marked pathological phenomena.
The determination of the blood glucose provides the physician only with information regarding the metabolic state at the time of taking the blood sample. On the other hand, a long-term control regarding the metabolic state over the past 120 days is possible by determination of the glycosilated hemoglobin (HbA.sub.1). Precisely because of its half-life time, the measurement of serum fructosamine is appropriate for the determination of the metabolic control of diabetics by the maintenance of diet and by therapeutic measures going back over a medium period of time of about three weeks. Therefore, in conjunction with the established clinical diagnostic parameters of blood glucose, as well as glycosylated hemoglobin (HbA.sub.1), the diagnostic arsenal for the supervision of diabetics is extended by a valuable medium term parameter by means of a dependable, specific and practical method for the determination of the serum fructosamine.
A process for the determination of serum fructosamine, which in principle is easy to carry out, has been described by Baker in European Patent Specification No. 0,085,263 and by Johnson et al. in Clin. Chim. Acta 127, 87-95 (1982). This depends upon the fact that the ketoamine form passes over into an enol form in aqueous alkaline medium which acts reducingly on tetrazolium salts, for example nitrotetrazolium blue, and thereby provides a formazan colored material. The extent of the colored material formation measured photometrically within a definite interval of time at 37.degree. C. is said to be proportional to the amount of fructosamine present.
In the case of the use of serum as sample material, this test is subject to disturbance since natural serum components, such as bilirubin, uric acid and SH groups, also act reducingly on tetrazolium salts. Medicaments, for example, alpha-methyldopa, decomposition products of medicaments, for example gentisic acid which is a metabolite of acetylsalicylic acid, as well as ascorbic acid, also give rise to falsified measurement results, depending upon the concentration thereof in the serum.
Even in the case of otherwise inconspicuously normal and diabetic sera these disturbances have the effect that in the case of a method comparison with, for example, the furosine/HPLC method according to Schleicher and Wieland (J. Clin. Chem. Clin. Biochem. 19, 81-87 (1981)), which makes possible absolute indications regarding the extent of non-enzymatic glycosylation, and the process for the determination of serum fructosamine according to Baker, there is admittedly obtained a linear correlation of the fructosamine content. However, the Baker method indicates a considerable non-enzymatic glycosylation even when this is, in fact, certainly not the case. Schematically, in a graph in which the results of the Baker method are plotted against those of the furosine/HLPC method, this has the result that, in the case of the Baker test, even when serum fructosamine is completely absent, an axis intercept occurs, the value of which corresponds to about 50% of that of the average non-diabetic (cf. FIG. 1 of the accompanying drawings).
Furthermore, serum fructosamine determinations have hitherto suffered from disturbances which are due to the total protein content which varies from sample to sample. They give rise to variations of the measurement values and thereby reduce the sensitivity of the determination process. These disturbances are known as matrix effects. Such effects are especially noticeable in the case of the addition of further protein, such as is usually the case, for example, in the preparation of standard solutions. Increasing amounts of protein slow down the reaction between fructosamine and color reagent (cf. E. J. Hindle et al., Ann. Clin. Biochem. 22, 84-89 (1985)).
Further difficulties arise in the case of fructosamine determinations in hyperlipemic sera. In general, in order to obtain a measurement signal which is also sufficiently great even in the case of low fructosamine concentrations, a sample/reagent volume ratio of 0.1 is necessary. In the case of excessive triglyceride concentrations, however, with such a high proportion of sample, the resultant turbidity of the test batch has a negative effect in the case of a photometric measurement, the fructosamine determination being made considerably more difficult or even prevented.
Therefore, there is still a need for a process for the specific determination of the serum fructosamine content in blood or in samples obtained from blood which does not suffer from the above-mentioned disadvantages. Consequently, it is an object of the present invention to provide such a process.